(1) Summary of the Invention
The present invention relates to a method for testing for the risk of long-term complications of diabetes based upon expression of genetic material encoding aldose reductase in cells. In particular the present invention relates to an in vitro method which tests the cells in the presence of pathophysiologic levels of glucose for the test. Further, the present invention provides an in vitro method for determining whether a particular aldose reductase inhibitor may be effective against diabetes in a particular patient. Further still, the present invention provides a method for inhibiting aldose reductase expressing genetic material using a vector by delivering DNA or RNA which binds the genetic material in the cells to prevent the expression of aldose reductase.
(2) Description of Related Art
The prior art has recognized that there is a need for an effective method for testing patients for risk of diabetic complications at a cellular level. In general, there has been no method which is available.
There has been extensive research with aldose reductase which is involved in the complications of diabetes; however, no test has been developed for predicting the long-term complications of diabetes. The exact mechanism by which the enzyme is activated to abnormal levels has not been determined.
The success of a Diabetes Control and Complications Trial (DCCT) in 1993 rested in the convincing demonstration that sustained elevated levels of blood glucose increased the risk for the development of long-term complications of diabetes (N Eng J Med. 329:977-986 (1993)). Unanswered by the DCCT was the question why some individuals with elevated blood glucose levels were resistant to the development of these complications while others had excellent blood glucose control but a rapid clinical course to retinopathy, neuropathy or nephropathy. Pathways of glucose metabolism whose flux varies as a function of prevailing hyperglycemia are generally invoked as putative mediators of those chronic diabetic complications whose incidence and/or prevalence most closely parallels the duration and severity of antecedent hyperglycemia. But metabolic pathways, such as the polyol pathway, are rarely invoked to explain the wide variation of diabetic complications among patients with similar degrees of chronic hyperglycemia.
The polyol hypothesis asserts that diabetic complications result, in part, from the direct or indirect consequences of sorbitol production from excess glucose by aldose reductase (AR). Aldose reductase has been thought to be implicated in the development of long-term complications of diabetes for more than a quarter century, yet little is known about the pathogenetic determinants of AR expression in the normal population, subjects with diabetes and the presence or absence of long-term complications of the disease. Recent evidence has linked increased tissue-specific catalytic capacity or protein content AR enzymes with the prevalence and/or severity of diabetic complications, but it is not clear whether this is a cause or an effect. In multiple clinical trials, AR inhibitors (ARIs) have had mixed success (or failure) in the treatment or prevention of long-term complications of diabetes. Nonetheless, part of this limited success with the use of ARIs may be due to the inability to prospectively identify those subjects who would benefit from their use prior to the establishment of irreversible end-organ damage from diabetes.
Heterogeneity of AR protein expression has been hypothesized for almost 12 years beginning with Srivastava's and Das's report of AR activation in human tissues (Srivastava, S. K., et al., Biochim Biophys Acta 800:220-227 (1984)). For more than ten years, a theory of heterogeneity of AR protein activity causing increased susceptibility to long-term complications of diabetes has been proposed (Srivastava, S. K., et al., Biochim Biophys Acta 870:302-311 (1986)). Studies have demonstrated that elevations of ambient glucose will increase AR activity in erythrocytes from both normal and diabetic subjects (Lyons, P. A., et al., Medical Research and Clinical Practice 14:9-14 (1991); Hamada, Y., et al., Diabetes 40:1233-1240 (1991); Ana, H. P., Central African Journal of Medicine 41(6):199-202 (1995); and Funasako, M., et al., Mechanisms of Aging and Development 73:137-143 (1994)) and increased AR protein activity has been associated with the presence or absence of diabetic complications in multiple studies using both erythrocytes or neutrophiles (Hamada, Y., et al., Diabetic Medicine 10:33-38 (1993); Aida, K., et al., Diabetes Care 13(5):461-467 (1990); Nishimura, C., et al., Diabetologia 37:328-330 (1994); and Dent, M. T., et al., Diabetic Medicine 8:439-442 (1991)).
The human AR gene has been identified (Graham, A., et al., J. Bio. Chem. 266(11):6872-6877 (1991)) and mapped to a single locus on the 7q35 human chromosome (Graham, A., et al., Hum Genet 86:509-514 (1991)). While a true polymorphism of the AR gene has never been reported, a restriction length polymorphism to an (A-C).sub.n dinucleotide repeat (microsatellite DNA 5' of the promoter) has been identified from a population of Chinese subjects with Type II diabetes and found to be associated with the presence of early-onset retinopathy but not nephropathy (Ben, D., et al., Diabetes 44:727-732 (1995)). One study has proposed the existence of a polymorphic site at the first intron (in the noncoding region of the AR gene) which was also associated with retinopathy but not nephropathy (Patell, A., et al., Enzymology and Molecular Biology of Carbonyl Metabolism 4. H. Weine, Ed. Plenum Press, N.Y. pp. 325-332 (1993)). These studies did not determine the levels of AR activity in the populations studied, hence it is not known if expression of an AR "polymorphism" was associated with changes in AR activity or sorbitol production. These human studies have not prospectively identified those subjects who developed complications. Increased steady state levels of AR mRNA have been associated with the presence of diabetic nephropathy but not with non-diabetic causes of nephropathy (Shah, V., et al., ASN 6(3):455 (1995)). Shah, et al. determined that AR/.beta.-actin mRNA ratios were more than 3 fold elevated in those subjects with Type I diabetes and diabetic nephropathy as compared with subjects with diabetes and absence of nephropathy. This study did not determine the levels of AR activity in the study populations. The use of mRNA to ascertain heterogeneity of gene expression for AR is necessitated by lack of understanding of the genetic determinants of AR expression. Until the regulatory regions of the aldose reductase gene are characterized, analysis of mRNA seems an appropriate system to measure differences in AR gene expression. Nonetheless, these findings do suggest that heterogeneity of AR gene expression exists, and that elevated expression of AR mRNA may be seen in those subjects with diabetic kidney disease (Vinores, S. A., et al., Diabetes 37:1658-1664 (1988)). It is not known if heterogeneity of AR gene expression is genetically determined or an "epi-genetic" phenomena, resulting from the effects of diabetes on AR expression.
Heterogeneity of AR gene expression in primary cultures of human retinal pigment epithelial cells (RPE) has been described (Henry, D. N., et al., J. Clin. Invest. 92(2):617-23 (1993); Stevens, J. M., et al., Am. J. Physiol. 265(3 pt 1):E428-38 (1993); and Henry, D. N., et al., J. Am. Soc. Neph 6(3):362 (1995)). The retinal pigment epithelium supports and nourishes the neuroretinal cells in vivo. RPE cells exhibit glucose-induced, ARI-sensitive physiological impairment manifested by deterioration of electroretinograms in vivo and decreased rod outer segment phagocytosis in vitro, that is prevented by aldose reductase inhibitors (ARIs) (Del Monte, M. A., et al., Diabetes 40:1335-1345 (1991)). Immunohistochemical data suggest that AR protein is more abundant in RPE cells of patients with background or proliferative retinopathy (Vinores, S. A., et al., Diabetes 37:1658-1664 (1988)). Heterogeneity of AR expression in RPE cell cultures parallels AR activity and sorbitol production, and is responsive to the use of aldose reductase inhibitors in vitro. Thus RPE cells constitute a pathophysiologically relevant model in which to assess the effects of AR gene expression and polyol metabolism (Del Monte, M. A., et al., Diabetes 40:1335-1345 (1991)).
Trevisan, et al., (Trevisan, R., et al., Diabetes 41:1239-46 (1992)) have reported that in subjects with IDDM and nephropathy, both peripheral blood erythrocytes and cultured skin fibroblasts demonstrate a greater amiloride-sensitive sodium influx (Na+/H antiport activity) and enhanced cell proliferation than they do in subjects with IDDM and without nephropathy. To determine whether the tendency for NIDDM to run in families could relate to genetically determined defects in insulin stimulation of glycogen synthesis, Wells, et al., cultured forearm skin fibroblasts from subjects with a strong family history of NIDDM and from subjects without any family history of NIDDM as control subjects. Rates of glycogen synthesis were lower in NIDDM subjects both with basal and maximal insulin stimulation (Wells, A. M., et al., Diabetes 41:583-589 (1993)). Okuda, et al. have reported the restoration of myo-inositol uptake by eicosapentaenoic acid in skin fibroblasts cultured in a high glucose medium, and that myo-inositol uptake into skin fibroblasts was dependent on an active Na/K ATPase activity (Okuda, Y., et al., Life Sciences (57)5:71-74 (1995)). Thus, the use of fibroblasts can be used to characterize the pathogenetic expression of AR and diabetes.
An osmotically induced enhancer of aldose reductase gene transcription was identified in the 5'-untranslated region of the gene. This fragment works with both the homologous and a heterologous (thymidine kinase) promoter and was capable of increasing AR gene transcription independent of position and orientation to the promoter (Shiro Maeda, Masaki Togawa, Douglas N. Henry, Douglas A. Greene, Paul D. Killen. Characterization of an Osmotically Activated Enhancer of Aldose Reductase Gene Transcription. J. Am. Society of Nephr. 5(3):3414 (1994)).
The molecular mechanism underlying increased AR mRNA, protein and activity in patients with diabetes remains enigmatic. Otherwise silent genetic mutations which lead to high constitutive AR gene expression or inducibility of AR by elevated ambient glucose may predispose these subjects to perturbations of cellular metabolism linked to the polyol pathway and explain (in part) the clinical heterogeneity of diabetic complications. The early, prospective identification of particular patients prone to diabetic complications would motivate and justify intensive prophylactic metabolic intervention by either intensified metabolic control and/or selected use of aldose reductase inhibitors. Aberrant constitutive, generalized, or tissue-specific over-expression of AR in some patients with diabetes mellitus, whether on a genetic or acquired basis, could constitute such a predisposing factor.